CN110484563B - Mammal cell combined expression vector, expression system, preparation method and application - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and discloses a mammalian cell combined expression vector, an expression system, a preparation method and an application thereof, wherein an ARE sequence, an IRES sequence, a WPRE sequence and a screening marker gene sequence ARE inserted into the expression vector, the ARE sequence is shown as SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3, the IRES sequence is shown as SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6, and the combined expression vector has the structure that: ARE-promoter-target gene-WPRE-IRES-BSR-PolyA. The vector utilizes the anti-suppression element to carry out the optimized combination of different elements of the vector, on one hand, the transgene silencing is overcome, and on the other hand, the target gene is efficiently, durably and stably expressed in host cells.

Description

Mammal cell combined expression vector, expression system, preparation method and application

Technical Field

The invention relates to a mammalian cell combined expression vector, and also relates to an expression system containing the expression vector, a preparation method and application thereof, belonging to the technical field of genetic engineering.

Background

The recombinant protein is a protein produced by constructing a target gene into an expression vector by using a genetic engineering technology and realizing target expression in a heterologous cell. The production of recombinant proteins mainly involves four major systems: prokaryotic protein expression, yeast protein expression, insect cell protein expression, and mammalian cell protein expression. Proteins with complex structures or glycosylation important for activity play a crucial role in recombinant protein production, and are the most commonly used expression systems in recombinant pharmaceutical protein production at present. Coli prokaryotic expression systems and lower eukaryotic expression systems such as yeast lack complex post-translational modification functions, recombinant protein drugs need to be expressed in higher mammalian cells for activity. Currently, chinese Hamster Ovary (CHO) cell expression systems are used in nearly 70%. However, the low expression level of the target gene caused by Transgene silencing (Transgene silencing) becomes a bottleneck technology of a CHO cell expression system, and in addition, the defects of long screening period of high-yield stable cells, high cell culture cost and the like exist, so that the production of recombinant protein medicines is severely restricted.

In recent years, around increasing the expression of recombinant proteins in mammalian cells, researchers have made many beneficial attempts, where vector optimization is an effective strategy, such as the selection of strong promoters for expression vector construction, the use of enhancers, nuclear matrix binding regions, or demethylation during transgenic manipulation. However, most of the current vector optimization focuses on functional identification and research of a certain element, for example, the invention patent with the publication number of CN104975018A discloses a novel enhancer and application thereof, the enhancer is particularly suitable for CHO cells, the expression level of exogenous proteins in animal cells is greatly improved, and the expression level is improved by about 2 to 3 times, for example, the invention patent with the publication number of 201410129979.2 discloses a novel animal cell expression vector containing MAR core fragments, plasmids of MAR core fragments with artificial transcription factor binding sites added at two ends are particularly suitable for the CHO cells, and compared with plasmids of MAR core fragments of nuclear matrix binding region elements with artificial transcription factor binding sites added at two ends, the expression level of exogenous proteins in animal cells is greatly improved, and the expression level is improved by 10 to 17 times. However, these elements are still not ideal in improving the expression level of the transgene, and in addition, there are matching between different elements of the expression vector, such as MAR sequence and different promoters, and there are differences in the expression level of the target gene in CHO cells (Ho SC, mariati, yeo J H, fang S G, yang Y. Impact of using different expression reagents and matrix attachment regions on recombinant protein expression level and stability in transformed CHO cells. Mol Biotechnology. 2015 57 (2): 138-44), so it is necessary to optimize the combination between different elements of the vector to screen out the high efficiency expression vector.

Disclosure of Invention

The invention aims to provide a mammalian cell combined expression vector, which can enable a target gene to be efficiently, stably expressed for a long time in a host cell.

Another objective of the invention is to provide a preparation method of the mammalian cell combination expression vector.

The invention further aims to provide a eukaryotic cell expression system of the mammalian cell combined expression vector, a preparation method and application.

In order to realize the purpose, the invention adopts the following technical scheme:

the invention provides a mammalian cell combined expression vector, wherein an anti-repressor element sequence (ARE) shown as SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO 3, an Internal Ribosome Entry Site (IRES) shown as SEQ ID NO 4, SEQ ID NO 5 or SEQ ID NO 6, a woodchuck hepatitides virus posttranscriptional regulatory element sequence (WPRE) shown as SEQ ID NO 7 and a screening marker gene sequence ARE inserted into the expression vector; the ARE sequence is located upstream of a promoter, the WPRE sequence is located downstream of a gene of interest, the selectable marker gene is located downstream of the IRES sequence, and the gene of interest is located between the promoter and the IRES sequence.

Further, the selection marker gene sequence is selected from the blasticidin resistance gene sequence (BSR) as shown in SEQ ID NO: 8.

Furthermore, the starting vector of the expression vector is pIRES-neo2 or pIRES-neo3.

The invention provides a preparation method of the mammalian cell combined expression vector, which comprises the following steps:

step a: performing PCR amplification on the EGFP, performing double enzyme digestion on an EGFP reporter gene amplification product and a starting vector respectively, recovering enzyme digestion fragments, and performing connection, transformation and identification to obtain an expression vector I;

step b: respectively carrying out double enzyme digestion on the ARE sequence and the expression vector I, recovering enzyme digestion fragments, connecting, transforming and identifying to obtain an expression vector II;

step c: seamlessly cloning the IRES sequence to an expression vector II to obtain an expression vector III;

step d: respectively carrying out double enzyme digestion on the WPRE sequence and the expression vector III, recovering enzyme digestion fragments, and carrying out connection, transformation and identification to obtain an expression vector IV;

step e: and seamlessly cloning the screening marker gene sequence to an expression vector IV to obtain a mammal cell combined expression vector V.

The invention provides a eukaryotic cell expression system comprising the mammalian cell combined expression vector.

The invention also provides a preparation method of the eukaryotic cell expression system, which comprises the following steps:

step a: inserting a target gene between the promoter and the IRES sequence of the combined expression vector to construct a recombinant expression vector;

step b: transfecting the recombinant expression vector into a host cell, and screening to obtain an expression system.

The host cell is selected from one of DG44, DXB11, CHO-K1, CHO-S and the like. Preferably CHO-S cells. The transfection method includes calcium phosphate method, electroporation method, lipofection method, etc., and electroporation method is preferred.

The application of the mammalian cell combined expression vector and the eukaryotic cell expression system in preparing the medicine containing the target protein.

Compared with the prior art, the invention has the beneficial effects that:

the combined expression vector of the invention is inserted with a sequence of a resistance repression element, a sequence of an inner ribosome entry site, a woodchuck hepatitis virus transcription regulation element and a blasticidin resistance gene (screening marker gene), and the structure of the expression vector is as follows: ARE-promoter-target gene-WPRE-IRES-BSR-polyA. The vector utilizes the anti-suppression element to carry out the optimized combination of different elements of the vector, on one hand, the transgene silencing is overcome, and on the other hand, the target gene can be efficiently, durably and stably expressed in host cells. Under the same condition, compared with an expression vector without an ARE sequence and before optimization, the combined expression vector can obviously improve the expression level of the foreign gene, and can be used for producing recombinant protein.

Drawings

FIG. 1 is a plasmid map of the starting vector pIRES-neo2 in example 1.

FIG. 2 is a plasmid map of the mammalian cell combination expression vector pIRES-4 in example 1.

Fig. 3 is a flow cytometry assay to detect stable expression levels of EGFP in different expression vectors.

Fig. 4 is a flow cytometry assay to detect the long-term expression levels of EGFP in different expression vectors.

FIG. 5 shows ELISA detection of hepatitis B vaccine expression levels in different expression vectors.

Detailed Description

The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified.

The plasmid mini-cartridges used in examples and experimental examples were purchased from Shikoku Biotechnology Ltd (Cat: CW 0500S); the seamless cloning kit is purchased from Biyuntian biology company (the product number is D7010S), and tool enzyme, culture medium and the like are all commercial products; pIRES-neo2, pEGFP-C1 plasmids were purchased from Clontech, USA. Cell lines, plasmid vectors, reagents, tool enzymes, and the like used in examples and test examples are commercially available.

Example one construction of a mammalian cell combinatorial expression vector

This example is the construction of a combined expression vector containing artificially synthesized ARE-1, IRES-1, WPRE, BSR sequences, comprising the following steps:

1.1 construction of expression vector pIRES-EGFP containing EGFP reporter Gene

(1) EGFP reporter gene amplification

An upstream primer P-F and a downstream primer P-R are designed according to an Enhanced Green Fluorescent Protein (EGFP) gene sequence (GenBank: U55763.1, bases 613 to 1410) of a pEGFP-C1 vector, not I enzyme cutting sites and EcoRI enzyme cutting sites are respectively introduced into the 5' ends of the primers, and the primer sequences are shown as follows (the underlines are enzyme cutting sites):

P-F：5′-CCGGCGGCCGCATGGTGAGCAAGGGCGAGGAG-3′；

P-R：5′-CTAGAATTCTTACTAGATCCGGTGGATCC-3′。

the EGFP gene was amplified using pEGFP-C1 vector as template and primer P-F, P-R. The PCR reaction system is shown in Table 1.

TABLE 1 PCR reaction System

PCR reaction system	Concentration of reagent	Final concentration	Volume (μ L)
				10×PCRbuffer	10×	1×	2.5
Primer P-F	10μmol/L	0.4μmol/L	1.0
				Primer P-R	10μmol/L	0.4μmol/L	1.0
dNTP	25μmol/L	200μmol/L	2.0
				Template DNA	100ng/μL	4.0ng/μL	1.0
Taq enzyme	5U/μL	0.1U/μL	0.5
				ddH 2 O	/	/	17

Reaction procedure: pre-denaturation at 95 ℃ for 3min; denaturation at 94 ℃ 40s, annealing at 58 ℃ 30s, and extension at 72 ℃ for 40s, with 4 cycles per annealing temperature; finally annealing at 55 deg.C for 1min for 30 cycles, and extending at 72 deg.C for 3min; storing at 4 ℃.

And (4) recovering the PCR amplification product by agarose gel electrophoresis, and purifying the PCR amplification product to be sent to a biological company for sequencing verification. The result shows that the amplified DNA fragment is completely consistent with the EGFP sequence published by GenBank.

(2) Constructing expression vector pIRES-EGFP containing EGFP sequence and starting vector pIRES-neo2

The PCR amplification product of the EGFP is cut by Not I and EcoRI (the correct sequence is verified by sequencing), the DNA of the original vector pIRES-neo2 is cut by Not I and EcoRI (the pIRES-neo2 plasmid map is shown in figure 1), the cut result is identified by agarose gel electrophoresis, and the cut EGFP sequence fragment and the DNA of the original vector pIRES-neo2 are recovered by gel.

The EGFP sequence is cut by the enzyme system: 10 XNE Buffer 3.1. Mu.L, not I/Bam HI (10U/. Mu.L) each 1.0. Mu.L, make up water to 30. Mu.L; the enzyme digestion conditions are as follows: the enzyme was cleaved at 37 ℃ for 3h.

The starting vector pIRES-neo2 has an enzyme cutting system as follows: 10 XNE Buffer 3.1. Mu.L, not I/Bam HI (10U/. Mu.L) enzyme each 0.5. Mu.L, 0.81. Mu.g/. Mu.L of pIRES-neo2 DNA 1.23. Mu.L, and make up water to 20. Mu.L. After mixing well, incubate at 37 ℃ for 3h.

And connecting the cut EGFP sequence fragment with the starting vector pIRES-neo2 by using T4 ligase.

The starting vector pIRES-neo2 is connected with a system: 10 mu.L of 2 Xquick Ligation Buffer, 200ng of pIRES-neo2 linear DNA, 87.2ng of the EGFP sequence fragment after digestion, 1 mu.L of 350U/. Mu.L T4 ligase, water to 20 mu.L, and Ligation overnight at 16 ℃.

The ligation products were added to competent bacterial suspension of Escherichia coli (E.coli) JM109 for transformation, 100. Mu.L of the transformed bacterial solution was inoculated on an LB solid plate containing ampicillin, cultured overnight at 37 ℃, and single colony was picked for subculture by shaking. Extracting bacterial plasmids, and cutting the bacterial plasmids by using Not I/Bam HI double enzymes, wherein the enzyme cutting system is as follows: 10 XNE Buffer 3.1. Mu.L, not I/Bam HI (10U/. Mu.L) enzyme each 0.5. Mu.L, 0.81. Mu.g/. Mu.L of pIRES-neo2 DNA 1.23. Mu.L, and make up water to 20. Mu.L. After mixing well, incubate at 37 ℃ for 3h. And (3) verifying by nucleic acid electrophoresis, wherein the size of the band with correct enzyme digestion is 720bp, selecting a plasmid with correct enzyme digestion identification, and performing sequencing verification to finally obtain a correct expression vector I, which is named pIRES-EGFP in the embodiment.

1.2 construction of expression vector pIRES-1 containing ARE-1 sequence

An ARE-1 sequence (shown as SEQ ID NO: 1) is designed and artificially synthesized, and is specifically handed over to the general biological gene (Anhui) Co., ltd. In order to facilitate cloning and ensure sequence integrity, when synthesizing an ARE-1 sequence, a GTCTCGCGA sequence is introduced into the 5' end, wherein GTC is a protective base, and TCGCGA is an NruI enzyme cutting site; AGCACGCGT is introduced at the 3' end, wherein AGC is used as a protective base for enzyme digestion, and ACGCGT is used as an MluI enzyme digestion site.

The synthesized ARE-1 sequence was double digested with NruI/MluI, while the pIRES-EGFP vector was double digested with NruI/MluI. And identifying the digestion result by agarose gel electrophoresis, and recovering the ARE-1 sequence fragment and pIRES-EGFP linear plasmid DNA after digestion by gel.

The double enzyme cutting system of the ARE-1 sequence is as follows: ARE-1 sequence 10. Mu.L (1. Mu.g/. Mu.L), 10 XNE Buffer 3.1. Mu.L, nruI/MluI (10U/. Mu.L) 1.0. Mu.L each, make up water to 30. Mu.L; the enzyme digestion conditions are as follows: the enzyme was cleaved at 37 ℃ for 3min.

The double enzyme cutting system of the pIRES-EGFP vector is as follows: pIRES-EGFP plasmid 5. Mu.L (1. Mu.g/. Mu.L), 10 XNE Buffer 3.1. Mu.L, nruI/MluI (10U/. Mu.L) each 0.5. Mu.L, make up water to 20. Mu.L; the enzyme digestion conditions are as follows: the enzyme was cleaved at 37 ℃ for 3min.

The digested ARE-1 sequence fragment and pIRES-EGFP linear plasmid DNA (molar ratio 5:1) were ligated at 25 ℃ for 5min using a ligation kit of NEB. The ligation product was added to E.coli JM109 strain competent cell suspension for transformation, 150. Mu.L of the transformed bacterial solution was inoculated onto an LB plate containing ampicillin, cultured overnight at 37 ℃ and single colony subcultured was selected. Extracting recombinant plasmids, carrying out double enzyme digestion (NruI/MluI) verification, and carrying out sequencing verification on the plasmids with correct enzyme digestion verification to obtain an expression vector II with correct construction, wherein the expression vector II is named pIRES-1 in the embodiment.

1.3 construction of expression vector pIRES-2 containing IRES-1 sequence (replacement of IRES sequence on pIRES-1 vector)

(1) Synthesis of IRES-1 sequence

IRES-1 sequence (shown as SEQ ID NO: 4) was designed and artificially synthesized, and was specifically delivered to GenBank (Anhui) Ltd.

(2) Construction of expression vector pIRES-2

Replacing IRES sequence nucleotide sequence of pIRES-1 vector with IRES-1 by seamless cloning kit to form new expression vector; carrying out double enzyme digestion verification on the new expression vector by SmaI/PacI enzyme, taking the vector with correct enzyme digestion verification, and carrying out sequencing verification to obtain an expression vector III with correct construction, wherein the expression vector III is named as pIRES-2 in the embodiment.

1.4 construction of expression vector pIRES-3 containing WPRE sequence

(1) Synthesis of WPRE sequences

WPRE sequence (shown as SEQ ID NO: 7) is designed and artificially synthesized, and is specifically handed over to GenBank (Anhui) Co., ltd. In order to facilitate cloning and ensure sequence integrity, when synthesizing a WPRE sequence, a CCGGAATTC sequence is introduced into a 5' end, wherein CCG is a protective base, and GAATTC is an EcoRI enzyme digestion site; 3' end is introduced with CTAGGATCC, wherein CTA is used as protective base for enzyme digestion, GGATCC is BamHI digestion site.

(2) Construction of expression vector pIRES-3

The synthesized WPRE sequence is cut by EcoRI/BamHI enzyme, and the pIRES-2 vector DNA is cut by EcoRI/BamHI enzyme. And (3) identifying the enzyme digestion result by agarose gel electrophoresis, and recovering the WPRE sequence fragment and pIRES-2 vector DNA after enzyme digestion by gel.

The enzyme cutting system of the WPRE sequence is as follows: 10 XM buffer2. Mu.L, 10U/. Mu.L each of EcoRI, bamHI enzyme 0.5. Mu.L, 1.289. Mu.g/. Mu.L of EGFP amplification product 0.78. Mu.L, and water to 20. Mu.L. After mixing well, incubate at 37 ℃ for 6h.

The pIRES-2 vector has an enzyme cutting system as follows: 10 XK buffer2. Mu.L, 10U/. Mu.L each of EcoRI and BamHI enzymes 0.5. Mu.L, 0.81. Mu.g/. Mu.L of pIRES-2 DNA 1.23. Mu.L, and make up water to 20. Mu.L. After mixing well, incubate at 37 ℃ for 3h.

And (3) connecting the WPRE sequence fragment after enzyme digestion with pIRES-2 vector by using T4 ligase.

The linker system of pIRES-2 vector is: 10 mu.L of 2 Xquick Ligation Buffer, 200ng of pIRES-2 linear DNA, 87.2ng of the EGFP sequence fragment after digestion, 1 mu.L of 350U/. Mu.L of T4 ligase, water addition to 20 mu.L, and Ligation overnight at 16 ℃.

The ligation product was added to a competent bacterial suspension of Escherichia coli (E.coli) JM109 for transformation, 100. Mu.L of the transformed bacterial solution was inoculated on an LB solid plate containing ampicillin, cultured overnight at 37 ℃ and single colony was picked for subculture by shake. Extracting bacterial plasmid, cutting the bacterial plasmid by EcoRI and BamHI, wherein the enzyme cutting system is as follows: 10 XK buffer2. Mu.L, 10U/. Mu.L each of EcoRI and BamHI enzymes 0.5. Mu.L, 0.81. Mu.g/. Mu.L DNA ligated vector 1.23. Mu.L, and make up water to 20. Mu.L. After mixing well, incubate at 37 ℃ for 3h.

And (3) verifying by nucleic acid electrophoresis, wherein the size of the band with correct enzyme digestion is 720bp, selecting a plasmid with correct enzyme digestion identification, and performing sequencing verification to finally obtain a correct expression vector IV, which is named pIRES-3 in the embodiment.

1.5 construction of expression vector pIRES-4 containing BSR sequence

(1) Synthesis of BSR sequences

BSR sequence (shown as SEQ ID NO: 8) is designed and artificially synthesized, and is specifically handed over to Universal Bio-Gene (Anhui) Inc.

(2) Construction of mammalian cell Combined expression vector pIRES-4

Inserting the artificially synthesized BSR sequence into a pIRES-3 vector by adopting a seamless cloning kit, and replacing a screening marker gene sequence on the pIRES-3 vector with a BSR sequence of a blasticidin resistance gene; carrying out double enzyme digestion verification on the new expression vector by SmaI/PacI enzyme, taking the vector with correct enzyme digestion verification, and carrying out sequencing verification to obtain an expression vector V with correct construction, wherein the expression vector V is named as pIRES-4 (a plasmid map is shown in figure 2). The structure of the mammalian cell combined expression vector obtained after inserting the target gene between the promoter and the IRES-1 sequence in the combined expression vector pIRES-4 is as follows: (ARE-1) -promoter-gene of interest-WPRE- (IRES-1) -BSR-PolyA.

EXAMPLE two construction of eukaryotic expression System

CHO cells at 37 ℃ 5% CO ₂ Under the condition, the culture is carried out in a DMEM medium containing 10% inactivated fetal bovine serum. CHO cells (3X 106/well) were seeded in 6-well plates. Transfection was performed when the cells reached approximately 90% confluence after 24h plating. Lipo 3000 (Lipofectamine 3000) is used as a transfection reagent, the combined expression vector pIRES-4 obtained in the first embodiment is transfected into CHO cells, after 24 hours of transfection, G418 with the concentration of 800 micrograms/mL is used for screening for 2 weeks to generate positive clones, the G418 with the concentration of 800 micrograms/mL is replaced by the G418 with the concentration of 400 micrograms/mL for stable culture for 1 month, and then the mammalian host cells CHO are obtained.

EXAMPLE three methods for expressing proteins derived from mammals

3.1 constructing a combined expression vector pIRES-4 containing the EGFP gene sequence and the nucleotide sequences of artificially synthesized ARE-1, IRES-1, WPRE and BSR, and the construction method is the same as the first embodiment.

3.2 transfection of the Combined expression vector pIRES-4 into mammalian host cells CHO, the transfection method was the same as in the mammalian host cells example two above.

3.3 culturing the CHO cell to express the target protein.

Test example 1

An ARE-2 sequence (shown as SEQ ID NO: 2) and an ARE-3 sequence (shown as SEQ ID NO: 3) ARE designed and artificially synthesized, and ARE specifically handed over to the general biological gene (Anhui) Co., ltd.

IRES-2 sequence (shown as SEQ ID NO: 5) and IRES-3 sequence (shown as SEQ ID NO: 6) were designed and artificially synthesized, and were specifically delivered to GenBank Biogene (Anhui) Inc.

Comparative example 1 of mammalian cell combination expression vector: referring to step 1.2 of the first example, the ARE-1 sequence of pIRES-1 vector was replaced with ARE-2 (shown in SEQ ID NO: 2) by using NruI/MluI double digestion to construct a new expression vector; carrying out double enzyme digestion verification on the new expression vector by using NruI/MluI enzyme, taking the vector with correct enzyme digestion verification, and carrying out sequencing verification to construct a correct expression vector II, which is named as pIRES-1A; the other steps are the same as those in the first embodiment.

Comparative example 2 of mammalian cell combination expression vector: referring to step 1.2 of the first example, the ARE-1 sequence of pIRES-1 vector was replaced with ARE-3 (shown in SEQ ID NO: 3) by using NruI/MluI double digestion to construct a new expression vector; carrying out double enzyme digestion verification on the new expression vector by using NruI/MluI enzyme, taking the vector with correct enzyme digestion verification, and carrying out sequencing verification to construct a correct expression vector II, which is named as pIRES-1B; the other steps are the same as those of the first embodiment.

Comparative example 3 of mammalian cell combination expression vector: referring to step 1.3 of the first example, IRES-1 sequence of pIRES-2 vector was replaced with IRES-2 (shown in SEQ ID NO: 5) using a seamless cloning kit to construct a new expression vector; carrying out double enzyme digestion verification on the new expression vector by SmaI/PacI enzyme, taking the vector with correct enzyme digestion verification, and carrying out sequencing verification to construct a correct expression vector III which is named pIRES-2A; the other steps are the same as those of the first embodiment.

Comparative example 4 of mammalian cell combination expression vector: referring to step 1.3 of the first example, IRES-1 nucleotide sequence of pIRES-2 vector was replaced with IRES-3 (shown in SEQ ID NO: 6) using a seamless cloning kit to construct a new expression vector; carrying out double enzyme digestion verification on the new expression vector by SmaI/PacI enzyme, taking the vector with correct enzyme digestion verification, and carrying out sequencing verification to construct a correct expression vector III which is named pIRES-2B; the other steps are the same as those of the first embodiment.

Comparative example 1 of mammalian host cells: the expression vector pIRES-1 in the second example of constructing the mammalian host cell was replaced with the expression vector pIRES-1A in the comparative example 1 of the mammalian cell combination expression vector, and the other examples were the same as the second example. The structure of the mammalian cell combined expression vector obtained after inserting the target gene between the promoter and the IRES-1 sequence is as follows: (ARE-2) -promoter-Gene of interest-WPRE- (IRES-1) -BSR-PolyA.

Comparative example 2 of mammalian host cells: the expression vector pIRES-1 in the second example of constructing the mammalian host cell was replaced with the expression vector pIRES-1B in the comparative example 1 of the mammalian cell combination expression vector, and the other examples were the same as the second example. The structure of the mammalian cell combined expression vector obtained after inserting the target gene between the promoter and the IRES-1 sequence is as follows: (ARE-3) -promoter-Gene of interest-WPRE- (IRES-1) -BSR-PolyA.

Comparative example 3 of mammalian host cells: the expression vector pIRES-2 in the second example of constructing the mammalian host cell was replaced with the expression vector pIRES-2A in the comparative example 1 of the mammalian cell combination expression vector, and the other examples were the same as the second example. The structure of the mammalian cell combined expression vector obtained after inserting the target gene between the promoter and the IRES-2 sequence is as follows: (ARE-1) -promoter-Gene of interest-WPRE- (IRES-2) -BSR-PolyA.

Comparative example 4 of mammalian host cells: the expression vector pIRES-2 in the second example of constructing the mammalian host cell was replaced with the expression vector pIRES-2B in the comparative example 1 of the mammalian cell combination expression vector, and the other examples were the same as the second example. The structure of the mammalian cell combined expression vector obtained after inserting the target gene between the promoter and the IRES-3 sequence is as follows: (ARE-1) -promoter-Gene of interest-WPRE- (IRES-3) -BSR-PolyA.

The expression vectors pIRES-1, pIRES-2, pIRES-3 and pIRES-4 obtained in example 1 and the comparative example 1~4 of the expression vector are transfected into CHO cells respectively by pIRES-1A, pIRES-1B, pIRES-2A, pIRES-2B, and the specific steps are as follows:

(1) Cell transfection

CHO finenessCell in 37 deg.C and 5% CO ₂ Under the condition, the culture is carried out in a DMEM medium containing 10% inactivated fetal bovine serum. CHO cells (3X 106/well) were seeded in 6-well plates. Cells reached approximately 90% confluence after 24h plating.

The test was divided into 10 groups: (1) a normal CHO cell group; (2) control group-transfection control vector pIRES-EGFP; (3) transfecting an expression vector pIRES-1; (4) transfecting an expression vector pIRES-1A; (5) transfecting an expression vector pIRES-1B; (6) transfecting an expression vector pIRES-2; (7) transfecting an expression vector pIRES-2A; (8) transfecting an expression vector pIRES-2B; (9) transfecting an expression vector pIRES-3; the expression vector pIRES-4 is transfected in red. Lipo 3000 (Lipofectamine 3000) is used as a transfection reagent, and each test group of vectors are transfected into CHO cells.

(2) Selection of stably transfected cell lines

After 24 hours of transfection, the vectors of each test group were transfected and cultured in CHO cells (the number of CHO cells is about 2X 106) in a 24-well plate (500. Mu.L/well), and when positive clones appeared in the (1) ~ (8) groups after 2 weeks of selection with G418 at a concentration of 800. Mu.g/mL, G418 at a concentration of 800. Mu.g/mL was stably cultured for 1 month instead of G418 at a concentration of 400. Mu.g/mL, and then the cells of each test group were collected and subjected to flow cytometry. (9) Positive clones appeared after screening for 1 week by blasticidin with the concentration of 15 μ g/mL (screening time is shortened by 1 week), replacing blasticidin with the concentration of 15 μ g/mL with blasticidin with the concentration of 10 μ g/mL for stable culture for 1 month, and collecting cells of each test group for flow cytometry detection.

(3) Effect on Stable expression of EGFP Gene

(1) After the polyclonal CHO cells obtained by the groups (9) - (8) through subculturing and screening by using a culture medium containing G418 with the concentration of 500 mug/mL and the groups (9) - (C) through passages by using blasticidin with the concentration of 10 mug/mL for 30 days, the cells of each test group are collected for flow cytometry, and the results are shown in FIG. 3.3, under the same conditions, compared with the average expression level of the control vector pIRES-EGFP, the pIRES-1, pIRES-1A, pIRES-1B, pIRES-2, pIRES-2A, pIRES-2B, pIRES-3 and pIRES-4 vectors are respectively increased by 2.87, 2.15, 1.76, 3.24, 2.65, 2.31, 3.74 and 4.58 times, the expression trend is pIRES-4 >pIRES-2A >pIRES-2B >pIRES-1A >, namely, the expression level of pIRES-4 is the highest, and is increased by 4.58 times compared with the original vector pIRES-EGFP. All data are statistical data from triplicate experiments.

(4) Effect on Long-term expression of EGFP

After screening the obtained polyclonal CHO cells for 30 days, continuously culturing for 180 days, collecting cells of each test group for flow cytometry detection, and analyzing the influence of different expression vectors on the long-term expression of the recombinant target gene, wherein the result is shown in figure 4.

As can be seen from FIG. 4, compared with the control vector pIRES-EGFP, only the expression stability of the EGFP genes of two expression vectors of pIRES-1B, pIRES-2A is lower than that of the control vector pIRES-EGFP in pIRES-1, pIRES-1A, pIRES-1B, pIRES-2, pIRES-2A, pIRES-2B, pIRES-3 and pIRES-4 vectors, and the stable expression of the EGFP genes can be improved to a certain extent by the remaining 6 vectors, wherein the expression of the EGFP genes of the pIRES-4 vectors is most stable and is up to 87.6%, and the retention rate is increased by 1.39 times compared with that of the control vector pIRES-EGFP. All data are statistical data from triplicates.

Test example 2

In this test example, the synthesis of hepatitis B vaccine gene (GenBank: V00867.1) was carried out by GenBank, inc., of Universal Bio-Gene, anhui, inc.

(1) Construction of recombinant expression vector containing hepatitis B vaccine target gene

The EcoRI/BamHI enzyme is used for double digestion of the artificially synthesized hepatitis B vaccine gene sequence, and the EcoRI/BamHI enzyme is used for double digestion of the expression vectors pIRES-EGFP, pIRES-1, pIRES-2, pIRES-3 and pIRES-4. And identifying the enzyme digestion result by agarose gel electrophoresis, and recovering the gene sequence fragments of the hepatitis B vaccine after enzyme digestion and the linear plasmid DNA of pIRES-1, pIRES-2, pIRES-3 and pIRES-4 by gel.

The double enzyme digestion system of the hepatitis B vaccine gene sequence is as follows: hepatitis B vaccine sequence fragment 10U L (1U g/. Mu.L), 10 x NE Buffer 2.13U L, ecoRI/BamHI enzyme (10U/. Mu.L) each 1.0U L, make up water to 30U L; the enzyme digestion conditions are as follows: the enzyme was cleaved at 37 ℃ for 3min.

The plasmid double enzyme cutting system is as follows: plasmid 15. Mu.L (1. Mu.g/. Mu.L), 10 XNE buffer 2.12. Mu.L, ecoRI/BamHI (10U/. Mu.L) 0.5. Mu.L each, make up water to 20. Mu.L; the enzyme digestion conditions are as follows: the enzyme was cleaved at 37 ℃ for 3min.

The digested hepatitis B vaccine gene sequence fragment and linear plasmid DNA (molar ratio 5:1) were ligated at 25 ℃ for 5min using a ligation kit of NEB. The ligation products were added to competent cell suspension of Escherichia coli (E.coli) JM109 strain for transformation, 150. Mu.L of the transformant was inoculated onto an LB plate containing ampicillin, cultured overnight at 37 ℃ and single colony subculture was selected. Extracting a recombinant expression vector, carrying out double enzyme digestion (EcoRI/BamHI) verification, carrying out sequencing verification on the vector with correct enzyme digestion verification, and finally obtaining a correct combined expression vector, wherein the combined expression vectors containing expression vectors pIRES-EGFP, pIRES-1, pIRES-2, pIRES-3 and pIRES-4 are named as pIRES-EH and pIRES-1H, pIRES-2H, pIRES-3H, pIRES-4H respectively.

(2) Cell transfection and selection of stably transfected cell lines

1) Cell transfection

CHO cells at 37 ℃ 5% CO ₂ Under the condition, the culture is carried out in a DMEM medium containing 10% inactivated fetal bovine serum. CHO cells (3X 106/well) were seeded in 6-well plates. Cells reached approximately 90% confluence after 24h plating. The experiments were divided into 6 groups: (1) normal CHO cell group; (2) control group-transfection control vector pIRES-EH; (3) transfecting a recombinant vector pIRES-1H; (4) transfecting a recombinant vector pIRES-2H; (5) transfecting a recombinant expression vector pIRES-3H; (6) transfecting a recombinant expression vector pIRES-4H. Using Lip3000 (Lipofectamine 3000) as transfection reagent, co-transfecting each test group of vectors into CHO cells.

2) Selection of stably transfected cell lines

The vector was co-transfected into CHO cells (number of CHO cells: about 2X 106) in a 24-well plate (500. Mu.L/well), and screened with G418 at a concentration of 800. Mu.g/mL for 2 weeks, and when positive clones appeared, G418 at a concentration of 400. Mu.g/mL was stably cultured for 2 weeks, then the amount of inoculated cells per mL was 50 to 60 ten thousand, the rotation speed of the shaker was set at 110 to 120 rpm/min, and the culture supernatant was collected on day 7 of suspension culture.

(3) Hepatitis B vaccine expression level detection

And (3) detecting the expression quantity of the hepatitis B vaccine by using an ELISA kit. The results are shown in FIG. 5. As can be seen from FIG. 5, the expression levels of the hepatitis B vaccine containing the combined expression vectors pIRES-EH, pIRES-1H, pIRES-2H, pIRES-3H, pIRES-4H were 9.21. Mu.g/d/106 cells, 24.04. Mu.g/d/106 cells, 28.73. Mu.g/d/106 cells, 33.62. Mu.g/d/106 cells, and 38.96. Mu.g/d/106 cells, respectively. Compared with a control group pIRES-EH, the expression level of the hepatitis B vaccine containing the combined expression vector pIRES-1H, pIRES-2H, pIRES-3H, pIRES-4 is improved by 2.61, 3.12, 3.65 and 4.23 times, namely the expression vector can obviously improve the expression level of the foreign protein in animal cells.

The procedures used in the examples and the experimental examples are not specifically defined, but are generally performed by conventional techniques in the art, for example, by referring to the Molecular cloning laboratory Manual (Sambrook J & Russell DW. Molecular cloning: a laboratory Manual, 2001) compiled by Sambrook et al, or the instructions provided by the manufacturer of the product.

The above-mentioned embodiments are merely preferred embodiments of the present invention, which are merely illustrative and not restrictive, and it should be understood that other embodiments may be easily made by those skilled in the art by replacing or changing the technical contents disclosed in the specification, and therefore, all changes and modifications that are made on the principle of the present invention should be included in the scope of the claims of the present invention.

SEQUENCE LISTING

<110> Hualan biological vaccine Co., ltd, new county medical school

<120> mammalian cell combined expression vector, expression system, preparation method and application

<130> 2019

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 2101

<212> DNA

<213> ARE-1

<400> 1

aggtgggtgg atcacccgag gtcaggagtt caagaccagc ctggccaaca tggtaaaacc 60

tcgtctctac taaaaaatac gaaaaattag ctggttgtgg tggtgcgtgc ttgtaatccc 120

agctactcgg gaggctgagg caggagaatc acttgaatct gggaggcaga ggttgcagtg 180

agctgagata gtgccattgc actccagcct gggcaacaga cggagactct gtctccaaaa 240

aaaaaaaaaa aaatcttaga ggacaagaat ggctctctca aacttttgaa gaaagaataa 300

ataaattatg cagttctaga agaagtaatg gggatatagg tgcagctcat gatgaggaag 360

acttagctta actttcataa tgcatctgtc tggcctaaga cgtggtgagc tttttatgtc 420

tgaaaacatt ccaatataga atgataataa taatcacttc tgacccccct tttttttcct 480

ctccctagac tgtgaagcag aaaccccata tttttcttag ggaagtggct acgcactttg 540

tatttatatt aacaactacc ttatcaggaa attcatattg ttgccctttt atggatgggg 600

aaactggaca agtgacagag caaaatccaa acacagctgg ggatttccct cttttagatg 660

atgattttaa aagaatgctg ccagagagat tcttgcagtg ttggaggaca tatatgacct 720

ttaagatatt ttccagctca gagatgctat gaatgtatcc tgagtgcatg gatggacctc 780

agttttgcag attctgtagc ttatacaatt tggtggtttt ctttagaaga aaataacaca 840

tttataaata ttaaaatagg cccaagacct tacaagggca ttcatacaaa tgagaggctc 900

tgaagtttga gtttgttcac tttctagtta attatctcct gcctgtttgt cataaatgcg 960

tttagtaggg agctgctaat gacaggttcc tccaacagag tgtggaagaa ggagatgaca 1020

gctggcttcc cctctgggac agcctcagag ctagtgggga aactatgtta gcagagtgat 1080

gcagtgacca agaaaatagc actaggagaa agctggtcca tgagcagctg gtgagaaaag 1140

gggtggtaat catgtatgcc ctttcctgtt ttatttttta ttgggtttcc ttttgcctct 1200

caattccttc tgacaataca aaatgttggt tggaacatgg agcacctgga agtctggttc 1260

attttctctc agtctcttga tgttctctcg ggttcactgc ctattgttct cagttctaca 1320

cttgagcaat ctcctcaata gctaaagctt ccacaatgca gattttgtga tgacaaattc 1380

agcatcaccc agcagaactt aggttttttt ctgtcctccg tttcctgacc tttttcttct 1440

gagtgcttta tgtcacctcg tgaaccatcc tttccttagt catctaccta gcagtcctga 1500

ttcttttgac ttgtctccct acaccacaat aaatcactaa ttactatgga ttcaatccct 1560

aaaatttgca caaacttgca aatagattac gggttgaaac ttagagattt caaacttgag 1620

aaaaaagttt aaatcaagaa aaatgacctt taccttgaga gtagaggcaa tgtcatttcc 1680

aggaataatt ataataatat tgtgtttaat atttgtatgt aacatttgaa taccttcaat 1740

gttcttattt gtgttatttt aatctcttga tgttactaac tcatttggta gggaagaaaa 1800

catgctaaaa taggcatgag tgtcttatta aatgtgacaa gtgaatagat ggcagaaggt 1860

ggattcatat tcagttttcc atcaccctgg aaatcatgcg gagatgattt ctgcttgcaa 1920

ataaaactaa cccaatgagg ggaacagctg ttcttaggtg aaaacaaaac aaacacgcca 1980

aaaaccttta ttctctttat tatgaatcaa atttttcctc tcagataatt gttttattta 2040

tttattttta ttattattgt tattatgtcc agtctcactc tgtcgcctaa gctggcatga 2100

t 2101

<210> 2

<211> 1586

<212> DNA

<213> ARE-2

<400> 2

tgagttgggg tcctaagcca gaagttaact atgctttcat atattcttgc aagtagaagt 60

acagtgttgg tgtaaattcc ccttagatgg atagctaagc ccagaggaaa taatggtaat 120

tggaaccata tgaccgtatg caattcatgt gcatatttat atcaagaaaa gaacattata 180

ggtcgggtga gaccctattt tgttctgaca atgtcatctg tatttacatg tctgtttcgg 240

gagtttggat gtcaagggat tctgtgctgg attgtaaagc atgtgcttct gcttgatgta 300

gctactcaat tttgtattct tgactaataa agtcataaac ataattcaac ctctgtgtgc 360

gtgctctcct tccattaatt tatactttag caaaaagtat tgaatgtgtg tgttatgtaa 420

caatttccta taaattatat taaatgattt attagcttta ttcaataaag ttttaagtgt 480

tttcttctat gactacatta tttgttaaca agaaatttct ttaactgaaa acttcaagga 540

agactatctg ggtaactctt tcaaaaagaa ttgtccctgt attttgggat tgaatatatt 600

aatttcttgt actgttttaa cagcacataa ttttacaaga caagccactt tttcaaagcc 660

tgcttctcct cccattttcc ctatctctgt gattgacacc tccaacccct gtagcctgcc 720

tctgctctct cttaaccagt cctactgata ctacttccta agtatttttc agccctgtcc 780

ttcctctcca tcatgatgga ttcacttcca gttgaaatcc ttatggtacc ctccctggat 840

tatggcagta atcagagagc tggtctcctt aactcaggat tcacttcttc tcatctgttg 900

ttcacagtga catcagaaag atattttaaa atgatgaact agaattaatt atataaaaca 960

cacatacaca cataaataat acttaaattt ttcaatgatg ttccaattat gtaaaatata 1020

atataggagg cactttatgt tctggcctca atctttcaat tcaaacttat ctcctgccac 1080

tatctccttt gaacattgta ttccagctac tttagaataa taataataca taatattcat 1140

agagcccttc ctgggttcct atcaccgtac aaaatacttc acatataaca tttaatcttt 1200

gacaacttta ttaggcatgc acaattatta tctatctata tatctatatc tatatatata 1260

aaatctatat tttatagata agaaaataga gggtaaaaac ttgccaaaat tacaaagctt 1320

agaagtgtag cagttgggat ttgaatctag gcatcctgcc tctatagtct acagtggctt 1380

tcttgtgcca aaagccttgc agttccctag acttaacatt tctcaaaatc tgtgtctttc 1440

acatgctctt ccaattgtct ggaaaatctt tcccaacctc agtctaactg tggtactcat 1500

gttcacccca caagaattga ctccatctgt cccctctcca tgaaaatttc tttgaatctc 1560

agcactttgg gaggctgagg caggtg 1586

<210> 3

<211> 1031

<212> DNA

<213> ARE-3

<400> 3

gatcaagaaa gcactccggg ctccagaagg agccttccag gccagctttg agcataagct 60

gctgatgagc agtgagtgtc ttgagtagtg ttcagggcag catgttacca ttcatgcttg 120

acttctagcc agtgtgacga gaggctggag tcaggtctct agagagttga gcagctccag 180

ccttagatct cccagtctta tgcggtgtgc ccattcgctt tgtgtctgca gtcccctggc 240

cacacccagt aacagttctg ggatctatgg gagtagcttc cttagtgagc tttcccttca 300

aatactttgc aaccaggtag agaagtttgg agtgaaggtt ttgttcttcg tttcttcaca 360

atatggatat gcatcttctt ttgaaaatgt taaagtaaat tacctctctt ttcagatact 420

gtcttcatgc gaacttggta tcctgtttcc atcccagcct tctataaccc agtaacatct 480

tttttgaaac cagtgggtga gaaagacacc tggtcaggaa cgcggaccac aggacaactc 540

aggctcaccc acggcatcag actaaaggca aacaaggact ctgtataaag taccggtggc 600

atgtgtatta gtggagatgc agcctgtgct ctgcagacag ggagtcacac agacactttt 660

ctataatttc ttaagtgctt tgaatgttca agtagaaagt ctaacattaa atttgattga 720

acaattgtat attcatggaa tattttggaa cggaatacca aaaaatggca atagtggttc 780

tttctggatg gaagacaaac ttttcttctt taaaataaat tttattttat atatttgagg 840

ttgaccacat gaccttaagg atacatatag acagtaaact ggttactaca gtgaagcaaa 900

ttaacatatc taccatcgta catagttaca tttttttgtg tgacaggaac agctaaaatc 960

tacgtattta acaaaactcc taaagacaat acatttttat taactatagc cctcatgatg 1020

tacattagat c 1031

<210> 4

<211> 621

<212> DNA

<213> IRES-1

<400> 4

ttaaaactgg gagtgggttg ttcccactca ctccacccat gcggtgttgt actctgttat 60

tacggtaact ttgtacgcca gtttttccca cccttcccca taatgtaact tagaagtttg 120

tacaatatga ccaataggtg acaatcatcc agactgtcaa aggtcaagca cttctgtttc 180

cccggtcaat gaggatatgc tttacccaag gcaaaaacct tagagatcgt tatccccaca 240

ctgcctacac agagcccagt accatttttg atataattgg gttggtcgct ccctgcaaac 300

ccagcagtag acctggcaga tgaggctgga cattccccac tggcgacagt ggtccagcct 360

gcgtggctgc ctgctcaccc ttcttgggtg agaagcctaa ttattgacaa ggtgtgaaga 420

gccgcgtgtg ctcagtgtgc ttcctccggc ccctgaatgt ggctaacctt aaccctgcag 480

ccgttgccca taatccaatg ggtttgcggt cgtaatgcgt aagtgcggga tgggaccaac 540

tactttgggt gtccgtgttt cctgtttttc ttttgattgc attttatggt gacaatttat 600

agtgtataga ttgtcatcat g 621

<210> 5

<211> 580

<212> DNA

<213> IRES-2

<400> 5

aagaagaggt agcgagtgga cgtgactgct ctatcccggg caaaagggat agaaccagag 60

gtggggagtc tgggcagtcg gcgacccgcg aagacttgag gtgccgcagc ggcatccgga 120

gtagcgccgg gctccctccg gggtgcagcc gccgtcgggg gaagggcgcc acaggccggg 180

aagacctcct ccctttgtgt ccagtagtgg ggtccaccgg agggcggccc gtgggccggg 240

cctcaccgcg gcgctccggg actgtggggt caggctgcgt tgggtggacg cccacctcgc 300

caaccttcgg aggtccctgg gggtcttcgt gcgccccggg gctgcagaga tccaggggag 360

gcgcctgtga ggcccggacc tgccccgggg cgaagggtat gtggcgagac agagccctgc 420

acccctaatt cccggtggaa aactcctgtt gccgtttccc tccaccggcc tggagtctcc 480

cagtcttgtc ccggcagtgc cgccctcccc actaagacct aggcgcaaag gcttggctca 540

tggttgacag ctcagagaga gaaagatctg agggaagatg 580

<210> 6

<211> 221

<212> DNA

<213> IRES-3

<400> 6

ggtgaggtcg acgccggcca agacagcaca gacagattga cctattgggg tgtttcgcga 60

gtgtgagagg gaagcgccgc ggcctgtatt tctagacctg cccttcgcct ggttcgtggc 120

gccttgtgac cccgggcccc tgccgcctgc aagtcggaaa ttgcgctgtg ctcctgtgct 180

acggcctgtg gctggactgc ctgctgctgc ccaactggct g 221

<210> 7

<211> 621

<212> DNA

<213> WPRE

<400> 7

gacctcgagg gaattccgat aatcaacctc tggattacaa aatttgtgaa agattgactg 60

gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt 120

atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc 180

tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt 240

ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga 300

ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct 360

gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaagctga 420

cgtcctttcc atggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct 480

gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc 540

tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg 600

cctccccgca tcgggaattc g 621

<210> 8

<211> 399

<212> DNA

<213> BSR

<400> 8

atggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac ggctacaatc 60

aacagcatcc ccatctctga agactacagc gtcgccagcg cagctctctc tagcgacggc 120

cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc agaactcgtg 180

gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt cgcgatcgga 240

aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct tctcgatctg 300

catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc agttgggatt 360

cgtgaattgc tgccctctgg ttatgtgtgg gagggctaa 399

Claims (7)

1. A mammalian cell combined expression vector is characterized in that an ARE sequence shown as SEQ ID NO. 1, an IRES sequence shown as SEQ ID NO. 4, a WPRE sequence shown as SEQ ID NO. 7 and a screening marker gene sequence ARE inserted into the expression vector; the ARE sequence is positioned at the upstream of a promoter, the WPRE sequence is positioned at the downstream of a target gene, the screening marker gene is positioned at the downstream of the IRES sequence, the target gene is positioned between the promoter and the IRES sequence, and the expression vector structure is ARE-promoter-target gene-WPRE-IRES-BSR-PolyA; the target gene is hepatitis B vaccine gene; the BSR is the blasticidin resistance gene.

2. The mammalian cell-associated expression vector of claim 1, wherein the selectable marker gene sequence is selected from the BSR sequence as set forth in SEQ ID NO. 8.

3. The mammalian cell-associated expression vector of claim 1, wherein the starting vector of the expression vector is pIRES-neo2 or pIRES-neo3.

4. A method of making a mammalian cell-associated expression vector according to any one of claims 1~3 comprising the steps of:

step a: performing PCR amplification on the EGFP, performing double enzyme digestion on an EGFP reporter gene amplification product and a starting vector respectively, recovering enzyme digestion fragments, and performing connection, transformation and identification to obtain an expression vector I;

step b: respectively carrying out double enzyme digestion on the ARE sequence and the expression vector I, recovering enzyme digestion fragments, connecting, transforming and identifying to obtain an expression vector II;

step c: seamlessly cloning the IRES sequence to an expression vector II to obtain an expression vector III;

step d: respectively carrying out double enzyme digestion on the WPRE sequence and the expression vector III, recovering enzyme digestion fragments, and carrying out connection, transformation and identification to obtain an expression vector IV;

step e: and seamlessly cloning the screening marker gene sequence to an expression vector IV to obtain a mammal cell combined expression vector V.

5. A eukaryotic cell expression system comprising the mammalian cell combination expression vector of any one of claims 1~3.

6. A method for producing the eukaryotic expression system of claim 5, comprising the steps of:

step a: inserting a target gene between a promoter and an IRES sequence of the combined expression vector of any one of claims 1~3 to construct a recombinant expression vector;

step b: transfecting the recombinant expression vector into a host cell, and screening to obtain an expression system.

7. Use of the mammalian cell combination expression vector of any one of claims 1~3 and the eukaryotic cell expression system of claim 5 in the preparation of a medicament comprising a protein of interest.

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